The present invention relates generally to materials and methods for use in immunological procedures for quantitative detection and isolation of the hormone erythropoietin in fluid samples. More specifically, the invention relates to a monoclonal anti-erythropoietin antibody, Epoclonalan, produced by a novel tumor cell line ATCC CRL 8164 and to uses of Epoclonalan in diagnostic assays on human fluids and in procedures for obtaining large quantities of, and characterizing human erythropoietin.
Erythropoiesis, the production of red blood cells, occurs continuously throughout the human life span to offset cell destruction. Erythropoiesis is a very precisely controlled physiological mechanism enabling sufficient numbers of red blood cells to be available in the blood for proper tissue oxygenation, but not so many that the cells would impede circulation. The formation of red blood cells occurs in the bone marrow and is under the control of the hormone, erythropoietin.
Erythropoietin, an acidic glycoprotein of approximately 34,000 molecular weight, may occur in three forms: .alpha., .beta., and asialo. The .alpha. and .beta. forms differ slightly in carbohydrate components, but have the same potency, biological activity and molecular weight. The asialo form is an .alpha. or .beta. form with the terminal carbohydrate (sialic acid) removed. Erythropoietin is present in very low concentrations in plasma when the body is in a healthy state wherein tissues receive sufficient oxygenation from the existing number of erythrocytes. This normal low concentration is enough to stimulate replacement of red blood cells which are lost normally through aging.
The amount of erythropoietin in the circulation is increased under conditions of hypoxia when the number of red blood cells in the circulation is reduced. Hypoxia may be caused by loss of large amounts of blood through hemorrhage, destruction of red blood cells by over-exposure to radiation, reduction in oxygen intake due to high altitudes or prolonged unconsciousness, or various forms of anemia. In response to tissues undergoing hypoxic stress, erythropoietin will increase red blood cell production by stimulating the conversion of primitive precursor cells in the bone marrow into pro-erythroblasts, bone marrow cells which subsequently mature, synthesize hemoglobin and are released into the circulation as red blood cells. When the number of red blood cells in circulation is greater than needed for normal tissue oxygen requirements, erythropoietin in circulation is decreased.
Because erythropoietin is essential in the process of red blood cell formation, the hormone has potential useful application in both the diagnosis and the treatment of blood disorders characterized by low or defective red blood cell production. Prior attempts to obtain erythropoietin in good yield from plasma or urine, however, have proven relatively unsuccessful. Complicated and sophisticated laboratory techniques are necessary and generally result in the collection of very small amounts of impure and unstable extracts containing erythropoietin.
U.S. Pat. No. 3,033,753 describes a method for partially purifying erythropoietin from sheep blood plasma. After injecting phenyl hydrazine hydrochloride into sheep in an amount sufficient to produce severe anemia, the blood is withdrawn and centrifuged to separate the plasma from the red cell portion. The plasma is then dialysed and brought into contact with an anion exchange resin. This eluate is further dialysed and lyophilized and introduced to two cation exchange resins. A suspension of the solid material of the second cation exchange resin is buffered, agitated and suction filtered. Further dialysis and lyophilization yields low yields of a crude solid extract containing erythropoietin.
Initial attempts to isolate erythropoietin from urine yielded unstable, biologically inactive preparations of the hormone. U.S. Pat. No. 3,865,801 describes a method of stabilizing the biological activity of a crude substance containing erythropoietin recovered from urine. Apparently the presence of two enzyme activities, proteases and sialidases, acts to degrade the polypeptide and to remove the sialic acid terminal sugars from active erythropoietin. The patent discloses a method of removing enzyme activities from the preparation of erythropoietin by dissolving crude urinary erythropoietin in a phosphate-buffered-saline solution and adding sodium p-aminosalicylate. A solution of phenol, a phosphate buffer, and sodium p-aminosalicylate is equilibrated and combined with the feed solution. The resulting solution is extracted and dialyzed against a phosphate-buffered-saline solution. The crude preparation containing erythropoietin purportedly retains 90% of erythropoietin activity, and is stable.
Another method of purifying human erythropoietin from urine of patients with aplastic anemia is described by the applicant Goldwasser and his co-workers in Miyake, Kung, and Goldwasser, "Purification of Human Erythropoietin," J. Biol. Chem., Vol. 252, No. 15 (Aug. 10, 1977), pp. 5558-5564. This seven-step procedure includes ion exchange chromatography, ethanol precipitation, gel filtration, and adsorption chromatography, and yields a crude erythropoietin preparation with a potency of 70,400 units/mg of protein in 21% yield. The purified hormone has a single electrophoretic component in polyacrylamide gels at pH 9, in the presence of sodium dodecyl sulfate at pH 7, and in the presence of Triton X-100 at pH 6. Two fractions of the same potency and molecular size, by sodium dodecyl sulfate gel electrophoresis, but differing slightly in mobility at pH 9, are obtained at the last step of fractionation.
Other techniques utilized to obtain purified erythropoietin involve immunological procedures. A polyclonal antibody directed against erythropoietin is developed by injecting an animal, preferably a rat or rabbit, with human erythropoietin. The injected human erythropoietin is recognized as a foreign antigenic substance by the immune system of the animal and elicits production of antibodies against the antigen. Differing cells responding to stimulation by the antigenic substance produce and release into circulation antibodies slightly different from those produced by other responding cells. The antibody activity remains in the serum of the animal when its blood is extracted. While unpurified serum or antibody preparations purified as a serum immunoglobulin G fraction may then be used in assays to detect and complex with human erythropoietin, the materials suffer from a major disadvantage. This serum antibody, composed of all the different antibodies produced by individual cells, is polyclonal in nature and will complex with components in crude extracts other than erythropoietin alone.
There exists, therefore, a substantial need in the art for a relatively simple method of obtaining large quantities of pure, biologically active erythropoietin from plasma or urine. There also exists a need for materials useful in the accurate quantitative detection of erythropoietin in human fluid samples.